Modified collagen compound and method of preparation

ABSTRACT

Chemically-modified collagen is prepared by reacting native collagen with a di or tri-carboxylic acid halide, di or tri-sulfonyl halide, di or tri-anhydride, or di or tri-reactive active ester coupling agent. The reaction is done in a controlled manner so that the degree of cross-linking is limited. Any remaining lysine epsilon amino groups present in the coupled collagen product may be converted to ureido, β-malicamino carboxyamido or sulfonamido groups by isocyanate, epoxy succinic acid, acid halide, anhydride, sulfonyl halide or active ester aminemodifying agents. The resultant soluble product when dissolved in a physiological buffer provides a viscoelastic solution having therapeutic application in a variety of surgical procedures, particularly in ophthalmic surgery. This viscoelastic solution &#34;melts,&#34; i.e., exhibits a dramatic loss of viscosity, when subjected to temperatures of between 32° and 48° C. The resultant insoluble product is suspended in physiological buffer and useful in tissue augmentation. The suspension containing the insoluble product exhibits a dramatic loss in viscosity at temperatures between about 40° and 70° C.

This is a continuation-in-part of application Ser. No. 104,777 filedOct. 5, 1987 and now U.S. Pat. No. 4,851,512 which is acontinuation-in-part of application Ser. No. 890,847 filed Aug. 6, 1986now U.S. Pat. No. 4,713,446 which is a continuation-in-part of Ser. No.773,310 filed Sep. 6, 1985, which is now abandoned.

FIELD OF THE INVENTION

This invention relates to a chemically-modified collagen compound, whichis adapted to be either soluble or insoluble in a physiological buffer.A soluble collagen compound of the present in a physiological buffersolution is useful: (a) as a anterior segment implant to maintainanterior chamber depth and to protect the corneal endothelium duringintracapsular and extracapsular cataract lens extraction and duringintraocular lens implantation; (b) as a surgical adjunct during cornealtransplant surgery to protect the corneal endothelium from contactingother ocular tissue and to prevent post-operative graft dislocation; (c)as a posterior segment implant during intraocular lens implantation andas an adjunct to retinal detachment surgery; and (d) as a vitreousreplacement. An insoluble collagen compound of the present invention ina physiological buffer suspension is useful in soft tissue augmentation.This invention also relates to the production of the collagen compoundby reacting purified, native, pepsin-treated collagen with anamine-reactive coupling agent and a monofunctional amine-reactivemodifying agent, either sequentially or simultaneously, in a controlledmanner so as to control the degree of coupling.

BACKGROUND OF THE INVENTION

Sodium hyaluronate, collagen gels and chondroitin sulfate solutions havebeen used in the anterior chamber to protect the corneal endotheliumfrom intraocular lens trauma and to maintain anterior chamber depth.Additionally, hyaluronate and collagen gels have been used as vitreousreplacements. None of these materials has proven to be ideal in suchapplications.

Chondroitin sulfate solutions do not exhibit pseudoplastic behavior,i.e., the viscosity is relatively constant at all shear rates.Accordingly, chondroitin sulfate solutions do not exhibit the samedegree of anterior chamber support as pseudoplastic fluids such as thoseprepared using sodium hyaluronate. Furthermore, since the viscosity ofthe chondroitin sulfate solutions does not decrease at increasing shearrates (as do pseudoplastic materials) extremely high pressures areneeded to apply or irrigate chondroitin sulfate solutions through asyringe (MacRae et al., "The Effects of Sodium Hyaluronate, ChondroitinSulfate, and Methyl Cellulose on the Corneal Endothelium and IntraocularPressure," American Journal of Ophthalmology, 95:332-341 (1983)).Additionally, commercially available chondroitin sulfate solutions (20to 50 percent solutions) have osmolarities in excess of 500 mOs_(m).Such high osmolarities are detrimental to the corneal endothelium.Lastly, as reported by MacRae et al. in the American Journal ofOphthalmology, supra, 20 percent chondroitin sulfate may cause a sharpincrease in intraocular pressure in the first one to four hours afterintracameral injection and, therefore, anterior chamber washout isindicated.

Stenzel et al. ("Collagen Gels: Design for a vitreous Replacement",Science 164: 1282-1283 (1969)), Dunn et al. ("Collagen-Derived Membrane:Corneal Implantation", Science, 157: 1329-1330 (1967)) and Rubin et al.("Collagen as a Vehicle for Drug Delivery", J. Clinical Pharmacology,Aug.-Sept., Pages 309-312 (1973)) have described the use of stabilizedcollagen membranes and gels to serve as drug delivery devices, vitreousreplacement gels and cornea transplants. Introduction of crosslinks wasaccomplished by heat, ultraviolet radiation or glutaraldehyde reaction.

U.S. Pat. No. 4,409,332 discloses membranes and gels composed ofcomplexes of reconstituted collagen with alkaline phosphatase,crosslinked with glutaraldehyde, UV radiation or gamma radiation. Thesecomplexes are said to be useful as vitreous replacements forophthalmologic therapy.

U.S. Pat. No. 4,164,559 describes a chemically-modified collagenmembrane which is useful as a carrier for ophthalmic medication. Thecollagen compounds disclosed are single collagen units which have beenacylated or esterified.

Collagen as an anterior chamber replacement is described by Kawakami("Operation for Aftercataract with the Injection of Collagen Gel intothe Anterior Chamber", Excerpta Medica International Congress Series,Vol. 2 (450), pages 1432-1434 (1975)). This investigation describes theinjection of ultraviolet crosslinked collagen gel into the anteriorchamber prior to extraction of the aftercataract.

The collagen gels described hereinabove have greater viscosities andthus afford more protection and support to eye tissues than doeschondroitin sulfate. However, known collagen gels are not pseudoplasticand fragment into small pieces when injected through a syringe.Additionally, collagen gels are generally hazy materials and have beenknown to cause inflammatory reactions in the anterior chamber and thevitreous (Advances in Vitreous Surgery, pages 601-623, Irvine andO'Malley, 1976).

Furthermore, collagen gels injected into the anterior chamber may causean elevation of intraocular pressure (Kawakami, E., "Operation forAftercataract with the Injection of Collagen Gel into the AnteriorChamber", supra).

Neither the chondroitin sulfate solutions nor the collagen gels used inophthalmic surgery are viscoelastic materials. Viscoelastic ophthalmicmaterials are preferred for several reasons. During surgery,viscoelastic materials protect cell and tissue surfaces from mechanicaltrauma; created space by separating two adjacent but not adherent tissuesurfaces, or by breaking normal or pathological tissue adhesions;maintain space allowing for safe surgical manipulations or by permittingthe insertion of implants without dislocating or touching sensitivetissues; contain hemorrhages; and also act as a "soft instrument" or"surgical tool" to move, manipulate or relocate tissues.

After surgery, viscosurgical materials may be used to retain space for adesired period of time, prevent or minimize postsurgical inflammationand localize bleeding, restrain fibrin coagulation, hold backinflammatory cells, and lubricate tissue surfaces which move relative toeach other and thereby prevent adhesion formation.

U.S. Pat. No. 4,141,973 discloses the use of highly-pure hyaluronic acidfor both vitreous and aqueous replacement. This material is colorless,transparent, nontoxic and viscoelastic. However, it too has a number ofdrawbacks. The most abundant natural source of hyaluronic acid isrooster combs. Due to the low yield from this source coupled with therelatively complicated process involved in extracting and isolating thiscompound, hyaluronic acid is an expensive product. Secondly, whilehyaluronic acid appears to be efficacious in reducing endothelial celldamage and in maintaining the anterior chamber during surgicalmanipulation, reports of elevated intraocular pressure, postoperatively,have been documented and it is recommended that this substance beremoved from the anterior chamber prior to closing the corneal incision(MacRae et al., "The Effects of Sodium Hyaluronate, Chondroitin Sulfateand Methyl Cellulose on the Corneal Endothelium and IntraocularPressure," supra). Lastly, hyaluronic acid does not adhere tointraocular lens surfaces or surgical instruments. By way of contrast,if one dips an intraocular lens into the viscoelastic collagen solutionof this invention, the solution adheres to the surface of theintraocular lens, thereby providing an increased degree of endothelialprotection.

SUMMARY OF THE INVENTION

The present invention provides a chemically-modified collagen compoundwhich comprises two or more native collagen molecules which are coupledat least one lysine epsilon amino group present on each collagenmolecule by a dicarbonyl, tricarbonyl, disulfonyl, or trisulfonylcoupling group, or a coupling group comprising a plurality of moieties,at least two or three of which are selected from the group consisting ofcarbonyl or sulfonyl groups. The carbonyl and/or sulfonyl groups presentin the coupling group are linked to teach other through saturated orunsaturated alkylene, arylene or mixed alkylene-arylene coupling chainshaving less than about twenty carbon atoms. The alkylene and/or arylenecoupling chains may contain heteroatoms, e.g., O, S or N, and may besubstituted in available aromatic positions by carboxyl groups, straightor branched chain alkyl groups of about 1 to 4 carbon atoms, straight orbranched chain alkoxy groups of about 1 to 4 carbon atoms, halogens andother non-reactive moieties, and in available aliphatic positions bycarboxyl groups and alkyl or alkoxy groups of about 1 to 4 carbon atoms.

More specifically, the coupling group has the general formula

    --B--A--B--

wherein B is independently CO, SO₂ or combinations thereof;

A is selected from any one of the following:

(1) an aromatic group having about 6 to 20 carbon atoms;

(2) ##STR1## wherein Ar is independently an aromatic ring having 6 to 10carbon atoms or a heteroaromatic ring containing atoms selected from thegroup consisting of C, N, O and S, and having about 5 to 10 atoms, orcombinations thereof;

J is hydrogen or --L--_(b) B wherein L is selected from the groupconsisting of phenylene, an alkylene of about 1 to 4 carbon atoms and anoxyalkylene of about 1 to 4 carbon atoms, b is 0 or 1, and B is asdescribed hereinabove, with the proviso that only one J is the chain maybe --L--_(b) B, in which case all other J's are hydrogen;

n is 0 or 1;

a is an integer between about 0 and 4; and

D is independently O, CO, S, SO, SO₂, ##STR2## --SO₂ --NH--,--NH--CO--NH--, wherein m is about 1 to 3, R is selected from the groupconsisting of phenyl, and a straight or branched chain alkyl or acylgroup having about 1 to 4 carbon atoms; and

J is as defined and restricted hereinabove;

(3) an aromatic group having about 6 to 10 carbon atoms, wherein saidaromatic group may be substituted in available positions by J wherein Jis as defined and restricted hereinabove;

(4) a heteroaromatic group containing atoms selected from the groupconsisting of C, N, O and S, and having from about 5 to 14 ring atoms,wherein said heteroaromatic group may by substituted in availablepositions by J, wherein J is as defined and restricted hereinabove;

(5) an aliphatic or arylaliphatic chain which contains one or twoolefinic or acetylenic groups and which contains about 2 to 20 carbonatoms, wherein said chain may be substituted in available positions byJ, wherein J is as defined and restricted hereinabove;

(6) an alicyclic ring which may be partially unsaturated, having about 3to 15 carbon atoms, wherein said alicyclic ring may be substituted inavailable positions by J wherein J is a defined and restrictedhereinabove;

(7) a heterocyclic ring which may be saturated or unsaturated and whichcontains atoms selected from the group consisting of C, N, O and S, andwhich has from about 5 to 12 ring atoms, wherein said heterocyclic ringmay be substituted in available positions by J wherein J is as definedand restricted hereinabove;

(8) ##STR3## wherein t is about 1 to 8;

E is independently O, NRJ, S, SO, SO₂, CO, ##STR4## CONH, SO₂ NH orNHCONH, wherein R is as defined hereinabove, m is about 1 to 3;

J is as defined and restricted hereinabove;

s is about 2 to 8;

p is about 0 to 4;

q is about 0 or 1; and

r is about 0 to 8, provided that when q is 1, r is greater than 0; and

(9) ##STR5## wherein G is independently an aromatic ring having about 6to 10 carbon atoms, or a heteroaromatic ring having about 5 to 10 atoms,or a heterocyclic ring having about 5 to 10 atoms, wherein theheteroaromatic and heterocyclic rings contain atoms selected from thegroup consisting of C, N, O and S;

J is as defined and restricted hereinabove;

w is about 1 to 8;

E and q are as defined hereinabove;

y is about 1 to 2; and

v is between about 0 and 4, provided that when q is 1, v is not 0.

Aromatic or heteroaromatic portions of A may be substituted in availablepositions by carboxyl groups, straight or branched chain alkyl groups ofabout 1 to 4 carbon atoms, straight or branched chain alkoxy groups ofabout 1 to 4 carbon atoms, halogens and other non-reactive moieties.Aliphatic, alicyclic and heterocyclic portions of A may be substitutedin available positions by carboxyl groups and straight or branched chainalkyl groups of about 1 to 4 carbon atoms.

The present invention also provides a chemically-modified collagencompound which comprises two or more native collagen molecules coupledas described above, wherein at least a protion of the remaining basicnitrogens present on the coupled collagen (principally amine groups) areconverted to ureido, β-malicamino carboxyamido or sulfonamido groups,which carboxyamide- or sulfonamido groups preferably contain at leastone carboxyl or sulfonic acid moiety, by isocyanate, epoxy succinc acid,acid halide, anhydride, sulfonyl halide or active ester amine-modifyingagents. That is, at least a portion of the uncoupled lysine epsilonamino groups present on the coupled collagen product are linked toamine-modifying groups, which amine-modifying groups are β-malic acidgroups or saturated or unsaturated alkane, arene or mixed alkane-areneN-substituted carbamoyl groups having 1-10 carbon atoms or sulfonamideor carboxamide groups having between about 2 and 20 carbon atoms, whichare terminated by one or two carboxylic or sulfonic acid moieties. Theamine-modifying groups may also contain up to about five heteroatoms,e.g., O, S and N, and may be substituted in available aromatic andaliphatic positions by carboxyl groups, alkyl or alkoxy groups of about1 to 4 carbon atoms, halogens and other non-reactive moieties. Morespecifically, the amine-modifying groups have the general formula:

    --B--A(B--K).sub.z

wherein z is 0 to 2, preferably, 1 or 2, B and A are as definedhereinabove with the proviso that J is hydrogen, and K is OH,N-substituted carbamoyl groups of the formula: ##STR6## where R is asubstituted or unsubstituted C₁₋₈ aliphatic, C₅₋₈ alicyclic, or C₆₋₁₀aromatic group, or β-malic acid groups of the formula: ##STR7## or itsmetal salts including Na⁺, K⁺, Li⁺, Cu⁺⁺, Fe⁺⁺, Fe⁺⁺⁺, Mg⁺⁺, and Al⁺⁺⁺salts.

The chemically-modified collagen compound of the present invention isprepared by coupling purified, pepsin-treated collagen to a limitedextent, accompanied by the modification of uncoupled basic nitrogens(principally amine groups) by a modifying agent which renders thesesites nonbasic, i.e., having a pKa of less than 4.

Applicants have found that the degree of coupling is highly important inproducing collagen solutions having viscoelastic, pseudoplasticproperties which allow them to be used successfully as aqueous orvitreous replacements in ophthalmic surgery. It has been found that ifthe coupling is too extensive, the product produced is not avisco-elastic solution, but is instead a collagen gel. Such a gel isuseful in applications such as graft tissue augmen- tation. Independentof the extent of coupling, a collagen gel is produced when the modifyingagent used is an isocyanate. It has also been discovered that if thecoupling is not extensive enough the solution will not be asviscoelastic as desired and will not possess the lubricative propertiesnecessary for an ophthalmic surgery aid. Applicants have also found thatit is necessary to render nonbasic most of the remaining uncoupled basicsites present on the coupled collagen molecules, preferably at the sametime introducing a negatively charged group, in order for the collagenproduct to resist fibrillogenesis.

The collagen solutions prepared according to the method of the presentinvention are found to be particularly useful in ophthalmic surgerysince:

(1) they are viscoelastic and possess lubricative properties whichprovide a degree of protection

(2) they are pseudoplastic and thus are easily injected through asyringe, yet have the ability to regain their original static viscosity;

(3) they are resistant to spontaneous fibrillogenesis, and thus retaintheir clear transparent nature, after insertion in the eye;

(4) they adhere to hydrophobic polymeric surfaces such aspolymethylmethacrylate or polypropylene intraocular lenses, and thus canbe used to coat such lenses to facilitate insertion into the anterior orposterior chamber of the eye;

(b 5) when injected into tissues, they decrease in viscosity and diluteinto the tissue fluid, leaving the site;

(6) they will not adversely increase intraocular pressure;

(7) they have low osmolarities of between about 200 to 400 mOs; and

(8) preferred embodiments are noninflammatory and biologicallycompatible.

Additionally, native collagen is available from a wide variety ofsources, e.g. bone, tendon, hide, etc. Accordingly, collagen is moreabundant and less expensive to obtain than tissue derived hyaluronicacid.

DETAILED DESCRIPTION OF THE INVENTION

The process by which the chemically-modified collagen compound isprepared comprises four major steps.

These steps are (not necessarily in this order):

I. Collection of Collagen Source Material;

II. Controlled Coupling of Collagen Source Material;

III. Modification of Remaining Uncoupled Basic Sites; and

IV. Collection, Purification and Reconstitution of Modified Collagen.

I. Collection of Collagen Source Material

The method of obtaining the collagen from the crude collagen source,e.g. tendon, hide, etc., is normally not critical, and some flexibilitymay be used in the selection of the particular tissue and the methodapplied thereto. Applicants prefer to extract collagen from a connectivetissue, such as bovine hide. If the collagen is to be used forophthalmic applications, it is preferred that it be obtained solely fromthe corium layer of the bovine hide, otherwise known as "split" hides.Split hides are commercially available from the Andre Manufacturing Co.,Newark, New Jersey.

The collagen may be solubilized by any of the standard extractionmethods, e.g. acid or salt extraction, enzyme-digestion, or acombination of these. Preferably, dehaired and cleaned hide issolubilized with a proteolytic enzyme (pepsin, for example) andsolubilized collagen is precipitated at pH 7, after inactivation andremoval of the enzyme, by addition of NaCl to about 2.5M. Pepsin-treatedcollagen precipitates leaving behind in solution (to be discarded) thedigested nonhelical terminal peptides of the collagen molecule and othernon-collagenous contaminates, e.g. saccharides, mucopolysaccharides,etc. Inactivated enzymes are removed by filtration and centrifugation at4° C. The pepsin-treated collagen is then further purified by repeatingredissolution in acidic water (pH 2-4) and reprecipitation by salttreatment, e.g. by the addition of 0.8M sodium chloride solution at pH3.

The purified collagen is preferably diafiltered using, for example, anAmicon DC-30 filtration system, commercially available from Amicon,Danvers, Mass. Preferably, a 0.1μmembrane filter is employed to filterout salts, proteins and other molecules having a molecular weight ofless than 300,000 daltons. Applicants have found that diafiltratiionincreases the transparency of the collagen product and may aid inreducing the incidence of aqueous flare. Additionally, if the collagenis to be used in surgical applications, it must be sterilized,preferably by filter sterilization techniques.

II. Controlled Coupling

The solubilized purified collagen molecules are coupled using couplingagents which have two or three groups which react with amines but do notreact with carboxyl groups. Such coupling agents include di- andtri-carboxylic acid halides, di- and tri-sulfonyl halides, di- andtri-anhydrides, di- and tri-reactive active esters and coupling agentscontaining at least two groups of the carboxylic acid halide, sulfonylhalide, anhydride or active ester type. Preferred aromatic and aliphaticdi- and tri-carboxylic acid halides include d-camphoric diacid cholride;4[p-(o-chlorocarbonylbenzoly)phenyl]butyryl chloride;furan-3,5-dicarboxylic chloride; fumaryl chloride; glutaryl chloride;succinyl chloride; sebacoyl chloride; isophthaloyl chloride;terephthaloyl chloride; 4-bromoisophthaloyl chloride; diglycolic diacidchloride; 1,1-cyclohexanediacetyl chloride; 2,2-dimethylglutarylchloride; thioglycolic acid dichloride; nitrilotriacetyl chloride;beta-methylcarballylic acid trichloride; hexadecanedioic aciddichloride; malonic acid dichloride; acetone dicarboxylic aciddichloride; oxydiacetyl chloride benzene-1,3,5-tricarbonyl chloride;4-chlorocarbonyl-phenoxyacetyl chloride; homophthaloyl chloride;4,4'-diphenyletherdicarboxylic acid dichloride;4,4'-diphenyulthioetherdicarboxylic acid dichloride;4,4'-diphenylsulfonedicarboxylic acid dichloride; acetylene dicarboxylicacid dichloride; cyclohexane-1,4-dicarboxylic acid dichloride;trans-3,6-endomethylene-1,2,3,6-tetrahydrophthaloyl chloride;4,4'-dithiodibutyryl chloride; diphenylmethane-4,4'-bis(oxyacetyl)chloride; N-(4-chlorocarbonylphenyl)anthranyloyl chloride;1,3-benzenebisoxyacetyl chloride; pyridine-3,5-dicarboxylic aciddichloride; pyridine-2,5-dicarboxylic acid dichloride;pyridine-2,4-dicarboxylic acid dichloride; pyrazine-2,3-dicarboxylicacid dichloride; and pyridine-2,6-dicarboxylic acid dichloride;ethyleneglycol bis-(4-chlorocarbonylphenyl)ether; diethyleneglycolbis-(4-chlorocarbonylphenyl)ether;bis-(4-chlorocarbonyl-2-tolyl)thioether; andN-chlorocarbonylmethyl-N-methylglutaramic acid chloride.

Preferred aromatic and aliphatic di- or trisulfonyl halides includepara-fluorosulfonylbenzenesulfonyl chloride; 1,3,5-benzenetrisulfonylchloride; 2,6-naphthalenedisulfonyl chloride; 4,4'-biphenyl disulfonylchloride; 1,10-decane-disulfonyl chloride; and4,4'-trans-stilbenedisulfonyl chloride.

Preferred di- and trianhydride coupling agents include1,2,4,5-benzenetetracarboxylic dianhydride; 3,4,9,10-perylenetetracarboxylic dianhydride; 3,3',4,4'-benzophenonetetracarboxylicdianhydride; 1,2,7,8-naphthalenetetracarboxylic dianhydride;pyromellitic dianhydride; 2,3,4,5-tetrahydrofurantetracarboxylic aciddianhydride; mellitic trianhydride; 1,2,3,4-cyclobutanetetracarboxylicdianhydride; bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylicdianhydride; cyclopentanetetracarboxylic dianhydride;ethylenediaminetetraacetic dianhydride; anddiethylenetriaminepentaacetic dianhydride.

Active esters are described by Greenstein and Wintz in "Chemistry of theAmino Acids", Vol. 2, John Wiley and Sons, Inc. (1961). Preferreddireactive active ester coupling agents include diphenyl succinate;bis(p-nitrophenyl) succinate; bis(cyanoethyl) glutarate; and di-S-phenyldithiosuccinate.

Preferred coupling agents containing combinations of amine-reactivegroups include 5-chlorosulfonyl-ortho-anisic acid chloride;2-chloro-5-fluorosulfonylbenzoyl chloride; 4-chlorosulfonylphenoxyacetylchloride; meta-fluorosulfonylbenzoyl chloride; and trimellitic anhydrideacid chloride.

The coupling agent is added to and mixed thoroughly with an aqueoussolution of the pepsin-treated collagen. Preferably, in order to limitthe degree of coupling, the reaction mixture contains purified collagenin a concentration of 0.05 to 0.3 percent by weight, and more preferably0.15 to 0.3 percent by weight.

The concentration of the coupling agent is dependent upon many factorsincluding the reactivity of the coupling agent. In general, however, theamount of the coupling agent is about 1 to 600 moles of coupling agentper mole of collagen, preferably about 50 to 500 moles of coupling agentper mole of collagen and more preferably about 100 to 200 moles ofcoupling agent per mole of collagen.

The pH of the reaction mixture is preferably maintained throughout thecoupling reaction at about 8 to 11, preferably at about 8.5 to 9.5, andmost preferably at about 9.0, by addition of a dilute base, e.g., lNsodium hydroxide. In this manner, almost all of the lysine epsilon aminogroups present on the collagen molecules are freed from their protonatedform, and become capable of reaction with either the coupling agent orthe modifying agent.

The coupling reaction is continued until substantially all, i.e., atleast 90 percent, of the coupling agent has either reacted with thecollagen or been hydrolyzed, normally about thirty minutes.

The degree and uniformity of the coupling reaction is dependent upon,and thus is controlled by the temperature, the solvent used to dissolvethe coupling agent, the rate of addition of the coupling agent, theidentity and form of the coupling agent, the concentration of thereactants, and the pH variations of the reaction mixture.

For example, some coupling agents are preferably added to the collagensolution as a solid. Applicants have found that by adding the couplingagent to the collagen as a solid, the degree of coupling can becontrolled. However, while addition of certain coupling agents in solidform is preferred, the coupling agent may be dissolved in a suitablesolvent before addition to the pepsin-treated collagen. Suitablesolvents are preferably water miscible and include N-methylpyrrolidone;N,N-dimethylformamide; acetone; ethylene glycol dimethyl ether; andacetonitrile. The particularly-preferred solvents have relatively highdielectric constants, i.e., greater than 25 and preferably greater than30 when measured at 25° C. Such particularly-preferred solvents includeN-methylpyrrolidone and N,N-dimethylformamide. Solvents with highdielectric constants offer another means of controlling the couplingsince they tend to limit the degree of coupling by promoting hydrolysisof the coupling agent. Alternatively, relatively water-immisciblesolvents may be used, giving a two phase reaction mixture. The use of atwo phase mixture limits the degree of coupling by limiting the numberof reaction sites to the surface of the solvent. An example of arelatively water-immiscible solvent is ethyl acetate. When a solution ofthe coupling agent in any solvent is used it is preferred that theamount of solvent be such that there is present about 0.5 to 10 ml ofsolvent per 100 ml of aqueous collagen solution and most preferablyabout 1 ml of solvent per 100 ml of collagen solution.

While the coupling reaction can be conducted at a temperature of betweenabout 0 and 35° C., Applicants have found that by allowing the couplingreaction to proceed at a temperature below about 20° C., preferablyabout 4° C., the reaction of the coupling agent with lysine epsilonamino groups present on the collagen can be encouraged.

The particularly-preferred couplers include succinyl chloride; glutarylchloride; terephthaloyl chloride;bicyclo-(2,2,2)-oct-7-ene-2,3,5,6-tetracarboxylic-2,3,5,6-dianhydride;1,2,4,5-benzenetetracarboxylic dianhydride;p-fluorosulfonylbenzenesulfonyl chloride; and 1,3,5-benzenetrisulfonylchloride, diethylene triamine pentaacetic dianhydride.

Applicants have found that with certain highly-reactive coupling agentssuch as terephthaloyl chloride;bicyclo-(2,2,2)-oct-7-ene-2,3,5,6-tetracarboxylic-2,3,5,6-dianhydride;1,2,4,5-benzenetetracarboxylic dianhydride; andp-fluorosulfonylbenzenesulfonyl chloride; or with solvent systems ofhigh dielectric constant and/or relatively high water miscibility, suchas N,N-dimethylformamide and N-methylpyrrolidone, the coupling reactionis preferably begun by premixing the reactants at an acidic pH of about3 to 5.5, before raising the pH to between 8 and 11 to affect reactionof the collagen amine groups with the coupling agent. By varying the pHin this manner, the coupling reaction is controlled to achieve thedesired viscoelastic product.

It is preferred, particularly with coupling agents of lesser reactivity,that the pH of the reaction mixture be increased to at least 11.5 at thecompletion of the coupling reaction in order to hydrolyze and unreactedportions of the coupling agent.

III. Modification of Remaining Basic Sites

The coupled collagen product contains reactive basic sites, principallyamine groups, which in order to produce a clear and transparent solutionfor ocular surgery must be chemically modified to provide a net neutralor preferably negative charge. Such modification of these reactive basicsites will enable the collagen product to resist fibril formation whenused in ophthalmic surgery. To this end the coupled collagen is reactedwith a monoreactive amine-modifying agent, also known as a monoacylatingor sulfonating agent. The modifying agent is preferably a compound, orcombination of compounds which contains an isocyanate, epoxy, acidic,carboxylic or sulfonic group or generates an acidic, carboxylic orsulfonic group during reaction. Preferably, the acid form of themodifying agent has a similar pKa to that of the hydrolyzed couplingagent in order to insure optimum precipitation of thechemically-modified collagen product. Useful modifying agents includeisocyanates, epoxy succinic acid, anhydrides, acid hlaides, sulfonylhalides and active esters. Isocyanate modifying agents tend to result ina modified collagen compound that is insoluble under physiologicalconditions. Useful isocyanates include C₁₋₈ alkyl isocyanates such asbutyl isocyanate, C₅₋₈ cycloalkyl isocyanates such as cyclohexylisocyanate, aryl and substituted aryl isocyanates such as phenylisocyanate, bromohexyl isocyanate, and 4-methoxyphenyl isocyanate, andaralkyl isocyanates such as benzyl isocyanate, and phenethyl isocyanate.Preferred anhydrides include cyclic anhydrides, such as glutaricanhydride; 3-ethyl-3-methylglutaric anhydride; alpha-2-carboxyethylglutaric anhydride; 3-methyglutaric anhydride; 2-phenylglutaricanhydride; dimethylglutaric anhydride; 1,8-naphthalic anhydride;4-chloro-1,8-naphthalic anhydride; 3,6-dinitro-1,8-naphathalicanhydride; 3-nitro-1,8-naphthalic anhydride; maleic anhydride;bromomaleic anhydride; dichloromaleic anhydride; succinic anhydride;S-acetyl mercaptosuccinic anhydride; 2,2,3,3-tetramethyl succinicanhydride; 2-dodecen-1-yl succinic anhydride; methyl succinic anhydride;citraconic anhydride; itaconic anhydride; 2,3-quinoxalinedicarboxylicanhydride; 1,2-cyclobutane dicarboxylic anhydride; diphenic anhydride;cyclohexane-4-methyl phthalic anhydride; homophthalic anhydride;tetrahydrophthalic anhydride; tetrachlorophthalic anhydride;tetrabromophthalic anhydride;1,4,5,6,7,7-hexachloro-5-norborene-2,3-dicarboxylic anhydride;3,6-endoxo-1,2,3,6-tetrahydrophthalic anhydride; 5-chloroisatoicanhydride; 3,4-pyridine dicarboxylic anhydride;carbobenzyloxy-L-glutamic anhydride; 1,2,4benzenetricarboxylicanhydride; o-sulfobenzoic anhydride; thiodiglycolic anhydride;2,3-pyridine dicarboxylic anhydride; 3-ketoglutaricanhydride-(1,3-acetone dicarboxylic anhydride); diglycolic anhydride;4-amino-1,8-naphthalic anhydride; and camphoric anhydride. Preferredsulfonyl chlorides include chlorosulfonylbenzenesulfonic acid. Preferredacid chlorides include sulfoacetyl chloride, the monoacid chlorides ofterephthalic acid and fumaric acid, and monomethyl succinate acidchloride. Preferred active esters include phenolates, such as monophenylterephthalate and cyanomethyl esters such as mono(cyanomethylsuccinate). Additionally, acylating agents such as benzoyl chloride,benzenesulfonyl chloride and butyrylchloride which do not producenegatively charged products may be used, but preferably in combinationwith the above modifiers.

The modification reaction is run in an aqueous medium so that completingreactions of acylation of the collagen amines and hydrolysis of themodifying agent occur simultaneously. As in the coupling reaction, theextent of each reaction depends on the pH, the percentage of remainingbasic sites on the coupled collagen, the temperature, and the nature andform of the modifying agent.

The modifying agent may be added to an aqueous solution of the coupledcollagen either neat or in a solvent. Suitable solvents are those whichare used to dissolve the coupling agent. Preferably the modifying agentis added as a solid or in a water-miscible solvent having a dielectricconstant less than about 25. When a solution of the modifying agent in asolvent is used it is preferred that the amount of solvent be such thatthere is present about 0.5 to 10.0 ml of solvent per 100 ml of aqueouscoupled collagen solution and most preferably about 1.0 ml of solventper 100 ml of collagen solution.

Preferably, a large stoichiometric excess of the modifying agent isadded to the collagen, due to the competitive hydrolysis of themodifying agent which occurs under the reaction conditions. The amountof modifying agent used must at least be sufficient to react with from60 to 100 percent of the unreacted lysine epsilon amino groups andpreferably enough to react with about 80 percent of the unreacted lysineepsilon amino groups. In as much as the hydrolysis of the modifyingagent becomes increasingly dominant as the percentage of lysine aminogroups declines, it is neither practical nor necessary to achieve 100percent reaction. The amount of modifying agent necessary to react withat least 80 percent of the amino groups is dependent upon the reactivityof the modifying agent and the particular solvent, if any. For example,active esters and sulfonyl chlorides favor reaction with amines overhydrolysis. Therefore, less of the sulfonyl chlorides and active estersare required for the modification reaction than would be required withother modifying agents. Normally, however, at least 100 moles ofmodifying agent per mole of initial purified collagen, preferably atleast 500 moles of modifying agent per mole of collagen and mostpreferably at least 750 moles of modifying agent per mole of collagen isrequired.

The reaction mixture is maintained throughout the reaction at a pH ofpreferably about 8 to 11, more preferably about 8.5 to 9.5, and mostpreferably about 9.0, by addition of a dilute base, e.g., lN sodiumhydroxide. The modification reaction is preferably continued for atleast thirty minutes in order to hydrolyze substantially all of anyunreacted modifying agent. As was the case for the coupling reaction,while the modification reaction can be conducted at a temperature ofbetween 0 and 35° C., it is preferably conducted at a temperature belowabout 20° C., preferably about 4° C., since reaction with amine groupsis favored over hydrolysis at lower temperatures.

In most cases the pH of the reaction mixture is increased to at least11.5 at the completion of the modification reaction to hydrolyze andunreacted modifying agent.

While what has been described thus far is a two-stage synthesis (firstcoupling of the collagen) followed by modification of the remainingunreacted basic sites) a variation of this procedure is to conduct boththe coupling and modification reactions simultaneously. In fact, withthe more reactive coupling agents including dianhydrides such as1,2,4,5-benzenetetracarboxylic dianhydride andbicyclo-(2,2,2)-oct-7-ene-2,3,5,6-tetracarboxylic-2,3,5,6-dianhydride;diacid halides such as succinyl chloride and 5-chlorosulfonyl-o-anisicacid chloride; and sulfonyl halides such as benzene trisulfonyl chlorideand p-fluorosulfonyl benzenesulfonyl chloride, simultaneously reactingthe collagen with the coupling agent and the modifying agent ispreferred in order to prevent an unacceptably high degree of coupling inthe resultant collagen product. When conducting the coupling andmodification reactions simultaneously, one-half of the modifying agent(i.e., at least 50 moles per mole of initial purified collagen) is addedto the reaction mixture, with the other half of the modifying agentbeing used to treat the coupled collagen product after coupling.

A still further variation for use with the more reactive coupling agentsis to pre-react the purified collagen with approximately one-fourth ofthe modifying agent (i.e., at least about 25 moles per mole of initialpurified collagen) prior to coupling. The remaining modifying agent isused to treat the collagen product after coupling.

IV. Modified Collagen Collection, Purification and Reconstitution

The modified collagen is precipitated by adjustment of the pH toward theisoelectric point. The precipitate is collected preferably bycentrifugation and is washed with sterile pyrogen-free water to removeany excess reagents. The purified chemically-modified collagen isreadied for use by reconstitution with enough physiological buffer andlN NaOH to yield a 0.1 to 7.5, preferably 0.5 to 5.0 percent by weightsolution of modified collagen at a pH of between 6.5 to 7.5, preferably7.0 to 7.4. Suitable physiological buffers contain NaCl and optionallyenough other salts such as KCl, CaCl₂, MgCl, CH₃ CO₂ Na, NaH₂ PO₄, Na₂HPO₄ and sodium citrate to provide the buffer with an osmolarity ofbetween about 200 and 400 mOs, preferably about 320 mOs. A preferredphysiological buffer contains 0.64 percent by weight NaCl, 0.075 percentby weight KCl, 0.048 percent by weight CaCl₂, 0.030 percent by weightMgCl₂, 0.39 percent by weight CH₃ CO₂ Na and 0.17 percent by weight C₆H₅ O₇ Na₃, and is commercially available as BSS™ from AlconLaboratories, Inc., Fort Worth, Texas. Preferably, for ophthalmicapplications the buffer should also contain phosphate salts, e.g., Na₂HPO₄ and NaH₂ PO₄. A more preferred phosphate buffered solution contains0.84 percent by weight NaCl, 0.054 percent by weight KCl, 0.017 percentby weight CaCl₂, 0.028 percent by weight Na₂ HPO₄ and 0.004 percent byweight NaH₂ PO₄.

A final preferred step is to filter the reconstituted collagen throughabout a 10 micron filter to remove any particulates which may haveaccumulated during processing. The filtered collagen is then preferablystored under a positive nitrogen pressure, at a temperature of about 4°C. to avoid contamination.

When the preferred chemically-modified collagen fractions preparedaccording to this invention are dissolved in enough physiological bufferto provide a 1 to 5 percent by weight collagen solution they are

(a) transparent and colorless with a transmission of about 100 percentfrom 400 to 700 nanometers and a refractive index approximately equal tothat of water or the aqueous humor, i.e., about 1.33-1.40;

(b) stable and collagen fiber free;

(c) viscoelastic, i.e., exhibit the Weissenberg Effect (Introduction toColloid and Surface Chemistry, London Butterworths, 1966);

(d) pseudoplastic, having lower viscosities at higher shear rates;

(e) thixotropic, i.e., recovers viscosity at rest after shear;

(f) noninflammatory and biocompatible, with an ability to be absorbedinto tissue fluid when injected into physiological tissue;

(g) possessing a melt temperature of from about 32° C. to about 48° C.;and

(h) possessing an osmolarity of about 260 to 340 mOs, preferably about280 to 320 mOs.

All of the properties (a) through (h) of the collagen solution relate toits therapeutic activity in ocular use.

(a) Transparency

That the preferred collagen solutions for ocular use, i.e., up to 5percent by weight chemically-modified collagen dissolved inphysiological buffer, are transparent, colorless and have refractiveindices approximately equal to the aqueous humor make them particularlyappropriate as aqueous or vitreous replacements during intracapsular andextracapsular cataract extraction, intraocular lens implantation,corneal transplantation, and repair of retinal detachment. Transparencyassures the surgeon that he/she can manipulate freely and maintain fullcontrol of the surgical procedure with complete and clear visibility inthe presence of any quantity of the viscoelastic collagen solution.

(b) Stability

Physiological stability is defined as resistance to spontaneousfibrillogenesis at pH 7.2 and a temperature of 32-42° C. Fibrillogenesisis defined as the self assembly of collagen molecules into insolubleaggregates. Collagen which has not been chemically-modified, as taughtby the present invention, so as to react substantially all free aminegroups is subject to spontaneous fibrillogenesis, for example, whendissolved in a physiological buffer and warmed to 37° C.nonchemically-modified collagen will spontaneously form a white opaquefibrous network. Resistance to fibrillogenesis means that the collagensolutions of the present invention will retain their clear transparentnature after insertion in the eye.

(c) Viscoelasticity

Collagen solutions of the invention exhibit what is known as theWeissenberg Effect, indicating that they are viscoelastic. TheWeissenberg Effect describes the tendency in viscoelastic solutions forflow to occur at right angles to an applied force. When a rotating rodis lowered into a Newtonian (nonviscoelastic) liquid, the liquid is setinto rotation and tends to move outwards, leaving a depression aroundthe rod. When the rotating rod is lowered into a viscoelastic liquid,the liquid may actually climb up the rod. The rotation of the rod causesthe liquid to be sheared circularly and, because of its elastic nature,it acts like a stretched rubber band tending to squeeze liquid intowards the center of the vessel and, therefore, up the rod.

The collagen solutions due to their viscoelastic character havelubricative properties which make them particularly useful as protectivecoatings on instruments and implants which are used near sensitivecellular and tissue surfaces. When used in the anterior chamber, theviscoelastic materials of this invention maintain anterior chamber depthand protect the corneal endothelium during intracapsular andextracapsular cataract lens extraction and during intraocular lensimplantation. Viscoelasticity is also important in vitreous surgery, inorder that the solution be able to push back the retina to its normalposition and not flow through the hole behind the retina. Furthermore,viscoelastic solutions provide long lasting support to the retina untilit is healed, and maintain the rheological properties of the vitreous.

(d) Pseudoplasticity

The collagen solutions prepared according to this invention showsignificant viscosity decreases when subjected to increasing shearrates. The steady state viscosity of 2 percent by weight solutions ofchemically-modified collagen in physiological buffer was measured usinga cone and plate viscometer (commercially available as a Model 605Mechanical Spectrometer from Rheometrics Co., Piscataway, New Jersey) ata temperature between about 19 and 24° C. and at a humidity of about 50percent. The steady state of viscosity was measured over a period ofabout 1.5 minutes. The viscosity of the collagen solutions was betweenabout 0.15×10⁶ and 4.0×10⁶ centipoise at a shear rate of 0.10 seconds⁻¹; between about 0.20×10⁵ and 7.5×10⁵ centipoise at a shear rate of 1.0seconds⁻¹ ; between about 0.3×10⁴ and 1.0×10⁵ centipoise at a shear rateof 10.0 seconds⁻¹ ; and between about 0.45×10³ and 2.0×10⁴ centipoise ata shear rate of 100.0 seconds⁻¹.

For ophthalmic applications a pseudoplastic material is ideal. At highshear stresses, i.e., during surgery when the eye tissues, instrumentsand/or implants are being manipulated within the eye, the viscosity ofthe material decreases thereby reducing the drag force on adjacenttissues, while at low shear stresses when the material is at rest theviscosity is high and the material acts as an effective lubricant forimplants and/or for tissue surfaces which move relative to each other.

Additionally, pseudoplasticity permits the surgeon to move the collagensolution with relative ease through small bore needles and into smalltissue spaces.

(e) Thixotropy

A thixotrophic liquid may be defined as a pseudoplastic material whichis able to regain its viscosity when allowed to rest for an extendedperiod of time after being stressed. In general the chemically-modifiedcollagen solutions of the present invention are able to regain theirsteady state viscosity after being injected through a syringe.Specifically, the collagen solutions regain 50 to 95 percent, preferably65 to 95 percent of their steady state viscosity within about sevenminutes after being sheared.

(f) Noninflammatory and Biologically Compatible

Preferred viscoelastic solutions of chemically-modified collagen inphysiological buffer (about 1 to 3 percent by weight) were evaluated asanterior chamber implants in several animal species including rabbits,canine, swine, geese, and cynomologous monkeys. The chemically-modifiedcollagen solutions were implanted in one eye and control materials suchas air, balanced salt solution, e.g., BSS™ or Healon™, commerciallyavailable from Pharmacia, where implanted in the contralateral eye. Bothtreated and control eyes were examined with a slit-lamp not more than 24hours after implantation and again at 24 hour intervals up to 2 weeks. Amodified McDonald-Shadduck score system (McDonald, T. O. and Shadduck,J. A. (1977). Eye Irritation. In: Advances in Modern Toxicology, Vol. 4,Dermatotoxicology and Pharmacology, pp. 162-166. New York: John Wiley &Sons, Inc.) was used to evaluate the eyes. This system includesevaluation and scoring of conjunctival congestion, swelling anddischarge, aqueous flare, iris inflammation, corneal cloudiness andedema, pannus formation, and anterior capsule appearance. In addition,evaluations of material present and extent of coverage in the anteriorchamber were also made. Such evaluation indicated the overallequivalence or superiority of the chemically-modified collagen with air,BSS™ and Healon™. Accordingly, the preferred collagen solutions weredetermined to be noninflammatory and biologically compatible.

(g) Melt Temperature

The melt temperature is that temperature at which the viscoelasticcollagen solution exhibits a dramatic loss of viscosity, i.e., theviscosity in centipoise decreases over 100 to 1,000 fold when measuredat a shear rate of 1 sec⁻¹. In general, the melt temperature of thecollagen solutions prepared according to the present invention,(measured using a differential scanning calorimeter) is between about32° C. and 48° C.

The melt temperature can be regulated by controlling the extent ofcoupling; a greater degree of coupling producing a material with ahigher melt temperature. Collagen solutions which have a melttemperature of between about 34° C. and 38° C. are most suitable asanterior chamber implants for use in cataract extraction, IOL surgeryand corneal transplants, and as viscoelastic surgical aids for cornealtransplants. Lower melt temperature materials are preferred in theseapplications so that the material will clear from the eye relativelyrapidly, i.e., within about twenty-four hours, thereby reducing thepotential for a transient increase in intraocular pressure. Materialshaving a higher melt temperature of between about 39° C. and 45° C. arepreferred in applications where a more thermally stable material isrequired, e.g., as a vitreous replacement, as a joint fluid replacementand as a viscoelastic surgical aid for corneal transplants.

(h) Osomlarity

The osmolarity of the collagen solution must not be so great or solittle as to produce osmotic trauma to cells which come in contact withthe solution. In general, the collagen solutions of the presentinvention are isotonic and have osmolarities of between about 200 and400 mOs, preferably between about 260 and 340 mOs and most preferablybetween about 280 and 320 mOs.

The preferred collagen solutions of the present invention haveparticular applicability in ophthalmic surgery as an aqueous or vitreousreplacement. The aqueous humor may be replaced by the collagen solutionafter various intraocular or extraocular surgical procedures in order toprevent cellular invasion of the anterior chamber, which would endangerthe regeneration and function of the iris, ciliary body and cornealendothelium. The preferred collagen solution may also be used as abiological prosthesis in the anterior chamber after cataract surgery inorder to push back prolapsed vitreous and, to provide separation betweenthe vitreous and cornea. Further, the collagen solution could be used inthe anterior chamber after keratoplasty to prevent adhesion formationbetween the corneal wound and the iris.

The preferred collagen solution may be implanted into the vitreous afterextensive intravitreal surgery (removal of hemorrhages, opacities, etc.)to prevent excessive cellular reaction, and development of fibrous bandsand preretinal tissue membranes.

Furthermore, the preferred collagen solutions of this invention areuseful in retinal detachment surgery to provide a viscoelastic tool inthe manipulation necessary for reattachment of the retina, to facilitatethe intraocular wound healing by preventing excessive fibrous tissueformation and development of intravitreal scar tissue.

The preferred viscoelastic solutions of the present invention adhere tohydrophobic polymeric surfaces such as polymethylmethacrylate orpolypropylene intraocular lenses. Thus, intraocular lenses can easily becoated with the collagen solution thereby causing less trauma andhazzard during insertion into the anterior or posterior chambers of theeye. The chemically-modified collagen could also be used as a wettingagent in contact lens solutions. Such a wetting solution would remain onthe lens for a longer time than previously known wetting solutions,thereby prolonging the comfort afforded in lens wearer.

The collagen solutions would have use as a vehicle for medication inophthalmic or orthopedic applications to prolong the effect of the drug.

Certain nonimmunogenic collagen solutions are useful in othertherapeutic applications to prevent fibrous tissue formation and theconsequent development of adhesion and scars. For example, in cases oftraumatic arthritis, osteoarthritis and bursitis it is contemplated thatnonimmunogenic collagen solutions can be used to replace the synovialfluid in a synovial space to impede the development of intraarticularfibrous tissue (pannus, ankylosis, adhesions) and to support the healingprocess of cartilage and synovial tissue. As used herein, the term"synovial space" is intended to mean that space which separates joints,tendons and/or bursae.

In anthroplasty, osteotomy and all types of intraarticular surgery, suchas arthroscopy, certain collagen solutions of the invention could beused to protect the articular cartilage surfaces from postoperativeinjury and from the possible harmful effect of prosthetic surfaces, toprevent excess fibrous tissue formation and to promote the normalhealing of the soft tissues and cartilage.

It is further contemplated that certain collagen solutions of thepresent invention could be implanted between tendons and their sheathsto minimize adhesion formation after any surgical procedure or aroundperipheral nerves and nerve roots after injury or surgery when damage tothe connective tissue around the nerve is extensive and excessive scarformation is expected. Implantation of the collagen solutions around thehealing (regenerating) nerve may protect it from invasion by connectivetissue cells.

In order to prevent adhesion formation between two endothelial orconnective tissue membranes, certain collagen solutions could beimplanted between mesothelial, pericardial and pleural sheets.

The chemically-modified collagen of the present invention when in a drymembrane form would also be useful as a wound dressing. The collagen canact as a barrier to water and microorganisms when used to cover skinwounds.

Certain collagen solutions could be used to separate tissue surfaces.The viscoelastic properties of the solution would protect the tissueduring surgical manipulation and postoperatively. The collagen solutionswould be beneficial in improving the gliding function of muscle sheathsand tendon sheaths in traumatic injuries.

In orthopedic or cardiovascular surgery certain collagen solutions wouldbe useful to lubricate and coat implants. Further, the solution could beused to prevent vascular grafts from contacting body fluids, and couldalso be used as a component of synthetic vessels.

Furthermore, the collagen solutions of the present invention would beuseful as moisturizers and lubricants in cosmetic creams and lotion.

Lyophilized, coupled collagen and coupled and amine-modified collagensolutions of this invention are useful as hemostatic agents.

Insoluble, coupled collagen having a pH of 7 and coupled andamine-modified collagen materials of the present invention can be usedfor soft tissue augmentation according to well known techniques. Forexample, a 1-2% by weight solids suspension of collagen according to thepresent invention can be injected under the dermis layer of the skin incosmetic surgery.

Other uses of the chemically-coupled and/or amine-modified collagencompounds and viscoelastic solutions of the invention will undoubtedlyoccur to those skilled in the art and thus, the foregoing descriptiondoes not limit the possible applications.

In order more clearly to disclose the nature of the present invention,the following examples illustrating compositions in accordance with theinvention and methods of using such compositions will now be described.It should be understood, however, that this is done solely by way ofexample and is intended neither to delineate the scope of the inventionnor limit the ambit of the appended claims.

EXAMPLE 1

Isolation and purification of collagen Type I was accomplished by thefollowing method. Clean dehaired bovine hide (200 g) was cryopulverizedand added to 15 liters 0.5M acetic acid solution at 4° C. The collagenwas allowed to solubilize for 1 hour. The terminal non-helical portionsof the telopeptide collagen molecules were cleaved from the helicalportions of the molecule by adding pepsin (5.86 g) to the collagensolution and agitating this mixture at 4° C. for 16 hours. The pH of thesolution was then increased to 7.0 by addition of lON sodium hydroxide.After 2 hours of mixing (while maintaining the temperature at 4° C.) thedenatured pepsin was removed from solution by filtration. The collagensolution was then made 2.5M in sodium chloride by gradual addition ofthe solid salt. The resultant collagen precipitate was collected andreconstituted in 0.5M acetic acid. The collagen was again precipitatedby addition of sodium chloride to 0.8M. The precipitate was collectedand reconstituted in 0.5M acetic acid. Precipitation of the collagen byaddition of sodium chloride to 0.8M was repeated. The precipitatecollected was reconstituted in 0.1M acetic acid to provide a high purity0.3 percent wt/wt collagen Type I solution having a pH of about 3.

The filter-sterilized purified collagen was chemically modified in thefollowing manner. All reactions were conducted under aseptic conditionsusing sterile solutions and reagents. The collagen solution (3000 ml)was treated with 5N sodium hydroxide at 4° C. to raise the pH to 9.0.Finely-divided succinic anhydride power (1.60 g) was added to thissolution. The solution was vigorously agitated and the pH was maintainedat 9.0±0.25 by gradual addition of lN sodium hydroxide. After about twominutes, succinyl chloride (0.60 g) was added. Agitation was continuedfor 30 minutes and the pH was maintained at 9.0±0.25 by addition of lNsodium hydroxide. The resultant coupled collagen product was furthertreated (at 24° C.) by addition of finely-divided succinic anhydride(1.60 g). As before, the pH was maintained at 9.0±0.25 by gradualaddition of lN sodium hydroxide. The solution was agitated for anadditional 60 minutes. The pH was decreased to 4.1 by addition of 6N HClin order to precipitate the chemically-modified collagen product. Theproduct was collected by centrifugation and washed successively withfour volumes of sterile water. The collagen precipitate was dissolved ina phosphate-buffered solution¹ to provide a 2 percent by weight modifiedcollagen solution and the pH was adjusted to 7.2 with lN sodiumhydroxide.

The collagen solution was colorless and transparent by both visualinspection and light/optical microscopy at 40x.

The chemically-modified collagen solution was evaluated as an anteriorchamber implant in New Zealand white rabbits, inbred beagle dogs,domestic white geese, Yorkshire pigs and cynomologous monkeys.Implantation was conducted as follows. The animal was anesthetizedintramuscularly with ketamine. After sedation, the orbital areas wereshaved and the animal was moved to surgery and anesthesized usinghalothane and nitrous oxide. The eyes were coated with chloromycetin andbetadine was applied to the surrounding areas. The eyes were lavagedwith BSS™. The orbital areas were allowed to dry and a speculum wasplaced in the eye. All surgical procedures were performed usingophthalmomicrosurgery.

An incision approximately 1 mm was made into the anterior chamber at thelimbus using a Supersharp Beaver™ Blade. Aqueous fluid drained and theaqueous chamber was irrigated with BSS™ or BSS™ containing heparin (1 ccof 5000 units/cc in 500 ml) and epinephrine (1 cc of 1:1000 in 500 ml).The anterior chamber was aspirated and completely deflated. The chamberwas then filled using a 27-gauge cannula with the chemically-modifiedcollagen (a 2 percent by weight solution in phosphate-bufferedsolution²). Contralateral control eyes were filled with BSS™, air, or asolution of viscoelastic sodium hyaluronate, commercially available asHealon™ from Pharmacia Co. The quantity of material injected into theanterior chamber was dependent upon the aqueous volume and the inherentintraocular pressure. Material was injected until back pressure forcedit out of the injection site.

Both treated and control eyes were evaluated by a slit-lamp microscopeusing the McDonald-Shadduck system at not more than 24 hours aftertreatment and again at 24 hour intervals up to 96 hours. This systemincludes evaluation and scoring of conjunctival congestion, swelling anddischarge, aqueous flare, iris inflammation, corneal cloudiness andedema, pannus formation and anterior capsule appearance. Such evaluationindicated overall superiority of the chemically-modified collagen madeaccording to this example to air, BSS™, and Healon™.

Chemically-modified collagen (a 2 percent by weight solution in aphosphate-buffered solution²) prepared according to this Example 1 wasevaluated as an anterior adjunct during intraocular lens implantation ina New Zealand white rabbit. It was observed that the collagen solutionmaintained inflation of the anterior chamber for at least 30 minutes andprovided excellent chamber depth suitable for extraction of the cataractlens and implantation of an intraocular lens (IOL). IOL insertion wasfacilitated while traumatic damage, as observed during IOL insertionwithout use of a viscoelastic material, was reduced. The collagenmaterial coated the surgical instruments and the surfaces of the IOL.

In order to test the biocompatibility of the modified collagen solutionthe following test was performed. Primary human endothelial cellcultures were maintained on multiwell plates coated with 1 percentgelatin in normal saline (0.9 percent NaCl). After reaching confluencythe cells were washed with normal saline and were flooded with thechemically-modified collagen solution (2 percent by weight inphosphate-buffered solution³). After one hour at 35.5° C. the collagensolution was aspirated and the cells were rinsed with serum-free media.The cells were incubated in the medium for an additional 24 hours. Thecell cultures were examined by phase contrast microscopy beforeapplication of the collagen solution, 3 minutes after application of thecollagen solution, one hour after application of the collagen and 24hours after removal of the collagen solution. The treated cells were ashealthy (i.e., had not died or undergone morphological changes) as thosewhich were untreated, indicating that the collagen solution had no toxiceffects.

The collagen solution (2 percent by weight in phosphate-bufferedsolution³) was shown to be viscoelastic, i.e., exhibit the WeissenbergEffect, using the following test. A motor-driven one-half inch impellerdiameter polyteetrafluoroethylene-coated stirring rod was inserted intoa 50 ml beaker containing about 30 ml of the collagen solution, and wasrotated at about 40 revolutions per minute. The collagen solution flowedat right angles to the applied force and moved up the stirring rod atleast 0.5 cm.

The viscosity of the collagen solution (2 percent by weight inphosphate-buffered solution³) was examined rheometrically as follows.Viscosity measurements were taken at room temperature (22-24° C.) with amechanical spectrometer (commercially available as a Model 605Mechanical Spectrometer from Rheometrics Co., Piscataway, New Jersey)using the cone and plate technique. The angle of the cone was maintainedat 0.1 radians, and the sample was sheared for 30 seconds to allowequilibration before the force was measured over a period of 1.5 minutesand the viscosity was determined. Viscosities were determined in thismanner at several shear rates and are reported in Table I.

                  TABLE I                                                         ______________________________________                                        Shear Rates   Viscosity                                                       (in sec.sup.-1)                                                                             (in centipoise)                                                 ______________________________________                                        100.0         4,215                                                           10.0          10,280                                                          1.0           65,200                                                          0.1           370,500                                                         ______________________________________                                    

The viscosity of the collagen solution decreases with increasing shearrates indicating that the solution is pseudoplastic.

The thixotropy of the collagen solution (2 percent by weight inphosphate-buffered solution⁴) was demonstrated as follows: Viscositymeasurements at a shear rate of 0.1 sec⁻¹ were determined using the coneand plate technique described above. After shear thinning at 500 sec⁻¹for 30 seconds, the shear force was removed and the sample was permittedto relax for 7 minutes. The sample was sheared again at a shear rate of0.1 sec⁻¹ and the viscosity again recorded. The results are reported inTable II.

                  TABLE II                                                        ______________________________________                                        Initial Viscosity                                                                          Viscosity After 7 Minutes                                        at 0.1 sec.sup.-1                                                                          Recovery at 0.1 sec.sup.-1                                                                    Percent                                          (in centipoise)                                                                            (in centipose)  Recovery                                         ______________________________________                                        370,500      266,760         72                                               ______________________________________                                    

The collagen solution (about 2 percent by weight in BSS™) was alsotested for efficacy as an anterior chamber replacement using thefollowing test, referred to hereinafter as the "syringe test." A 250microliter glass syringe barrel (Model 1725RN available from HamiltonCo., Reno, Nevada) was equipped with a plunger from the Model 1725Nsyringe also available from Hamilton Co. A brass weight was threaded tothe top of the plunger to exert a constant force of 64 grams. The teflontip of the plunger was gently abraded with 30 micron grit lapping paperuntil the plunger moved freely. The barrel was fitted with a 2-inch 22gauge removable needle., The collagen solution was introduced into thebarrel bubble-free by using a needleless 1 ml plastic tuberculinsyringe. The time to extrude 0.05 ml of solution, i.e. the time for theplunger to travel 0.5 in. down the barrel, was recorded in Table III.

                  TABLE III                                                       ______________________________________                                                  Collagen Concentration                                                                        Extrusion Time                                      Sample    (Percent by Weight)                                                                           (sec)                                               ______________________________________                                        1         2.06            20                                                  2         2.11            19                                                  3         2.24            31                                                  4         2.13            26                                                  ______________________________________                                    

Applicants have determined that in order to adequately maintain theanterior chamber of the eye an extrusion time greater than about 20 sec.is required. Additionally, in order for the solution to pass out of theeye an extrusion time of less than about 120 sec. is required.Preferably, the extrusion time is between about 20 and 40 seconds.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry was about 35 to 36° C.

EXAMPLE 2

Chemically-modified collagen is prepared according to the procedure ofExample 1 except that phthalic anhydride (2.35 g) is used in place ofsuccinic anhydride (1.60 g) wherever succinic anhydride was used.

The collagen solution is colorless and transparent as determined by bothvisual inspection and light/optical microscopy at 40_(x) .

The collagen solution (2 percent by weight in a phosphate-bufferedsolution⁵) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence or superiority of the chemically-modified collagen of thisexample with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in phosphate bufferedsolution⁵) exhibits the Weissenberg Effect when tested in accordancewith the procedure described in Example 1. The collagen solution ispseudoplastic, exhibiting decreasing viscosities at increasing shearrates when tested in accordance with the procedure of Example 1. Thecollagen solution is thixotropic, recovering about 65-95 percent of itsinitial viscosity within 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34° to 37° C.

EXAMPLE 3

Purified Type I collagen was prepared according to the proceduredescribed in Example 1 except that the purified collagen precipitate wasreconstituted in 1700 ml 0.05M acetic acid to provide a 0.15 percentwt/wt collagen solution. This solution was maintained at 4° C. andtreated with lON sodium hydroxide to raise the pH to 5. To thisvigorously agitated solution terephthaloyl chloride (0.1036 g) in 17 mlN,N'-dimethylformamide was added all at once. The pH of the stirringmixture was rapidly brought to 9.0±0.25 with 5N sodium hydroxide.Overshoot was corrected by back-addition of 6N HCl. Stirring wascontinued for 6 minutes while the pH was maintained at 9.0. The pH wasraised to 11.5 for 2 minutes and then returned again to 9.0 by additionsof 5NaOH and 6N, HCl, respectively in order to hydrolize the unreactedterephthaloyl chloride, thereby producing the coupled collagen product.

To the coupled collagen product maintained at 4° C., glutaric anhydride(0.1455 g) in 17 ml of acetone was added dropwise while stirring. The pHwas maintained at 9.0±0.25 with 1N sodium hydroxide. The solution wasagitated for an additional 14 minutes. The pH was taken to 11.8 with lNsodium hydroxide for 2 minutes and was then returned to 9.0 with 6N HClfor 15 minutes. The pH of the solution was then dropped to 4.1 byaddition of 6N HCl to precipitate the chemically-modified collagenproduct. The product was collected by centrifugation and washed as inExample 1. The modified collagen precipitate was dissolved in BSS™ toprovide a 2 percent by weight modified collagen solution, and the pH wasadjusted to 7.15 with lN sodium hydroxide.

This solution was colorless and transparent as determined by both visualinspection and light/optical microscopy at 40x.

Viscosity as a function of shear rate was measured in accordance withthe procedure described in Example 1, with the following results.

                  TABLE IV                                                        ______________________________________                                        Shear Rate (1/sec)                                                                             Viscosity (cp)                                               ______________________________________                                        100.0            2,750                                                        10.0             9,847                                                        1.0              61,490                                                       0.1              425,000                                                      ______________________________________                                    

Viscosity decreased as shear rate increased indicating that the 2percent by weight collagen solution was pseudoplastic.

Chemically-modified collagen (a 2 percent by weight solution in BSS™)was evaluated as an anterior chamber implant in New Zealand whiterabbits. The collagen solution maintained inflation of the anteriorchamber for at least 30 minutes and provided excellent chamber depthsuitable for extraction of the cataract lens and implantation of anintraocular lens (IOL). The collagen solution coated the surgicalinstruments and intraocular lenses.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution⁶) was evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicated overallequivalence or superiority of the chemically-modified collagen of thisexample with air, BSS™ and Healon™.

The biocompatibility of the collagen solution was tested as follows.Decontaminated human corneas having less than 5 percent cell death wereincubated for 4 days at 34° C. in media containing BSS™ collagensolution (2 percent by weight in BSS™) 25 percent by volume in BSS™ orcollagen solution (2 percent by weight in BSS™) 50 percent by volume inBSS™. After incubation the corneas were stained with trypan blue or atyaalizarin red and examined for cell morphology and density. Corneastreated with the collagen solutions were not significantly differentfrom corneas treated with BSS™.

The collagen solution (about 2 percent by weight in BSS™) exhibited theWeissenberg Effect when tested in accordance with the proceduresdescribed in Example 1.

The thixotropy of the 2 percent collagen solution was determined asdescribed in Example 1. The results are reported in Table V.

                  TABLE V                                                         ______________________________________                                        Initial Viscosity                                                                           Viscosity After 7 Min-                                          at 0.1 sec.sup.-1                                                                           utes Recovery at 0.1                                                                         Percent                                          (in centipoise)                                                                             sec.sup.-1 (in centipoise)                                                                   Recovery                                         ______________________________________                                        425,000       308,100        72                                               ______________________________________                                    

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry was about 39 to 40° C.

EXAMPLE 4

Chemically-modified collagen was prepared according to the procedure ofExample 3 except that diglycolic anhydride (0.124 g) in 17 ml of acetonewas used in place of glutaric anhydride as the amine modifier.

The collagen solution was colorless and transparent as determined byboth visual inspection and light/optical microscopy at 40x.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution⁷) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence or superiority of the chemically-modified collagen madeaccording to this example with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenbery Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65-95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 5

Chemically-modified collagen was prepared according to the procedure ofExample 3 except that 1,2,4-benzenetricarboxylic anhydride (0.425 g) wasused in place of the glutaric anhydride as the amine modifier. Afterprecipitation and washing, the collagen was dissolved in BSS™ to providea 2 percent by weight collagen solution. The pH of the solution wasadjusted to 7.2 with lN sodium hydroxide.

The collagen solution was colorless and transparent as determined byboth visual inspection and light/optical microscopy at 40x.

The collagen solution was evaluated as in an anterior chamber implant inNew Zealand white rabbits. The collagen solution maintained inflation ofthe anterior chamber for at least 30 minutes and provided excellentchamber depth suitable for extraction of the cataract lens andimplantation of an intraocular lens (IOL).

The collagen solution (2 percent by weight in BSS™) was evaluated as ananterior chamber implant in adult cats in accordance with the proceduredescribed in Example 1. The eyes were examined 24 and 48 hourspost-operatively by specular microscopy to evaluate general morphologyand density of endothelial cells. No adverse effects on the cornealendothelium were observed.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution⁸) was evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicated overallequivalence or superiority of the chemically-modified collagen of thisexample with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 75 percent of its initial viscosity within7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 35 to 36° C.

EXAMPLE 6

Chemically-modified collagen was prepared according to the procedure ofExample 3 except that cyclopentanetetracarboxylic dianhydride (0.1786 g)in 17 ml of N-methyl pyrrolidone was used in place of terephthaloylchloride as the coupler, and 1,2,4-benzenetricarboxylic anhydride(0.5667 g) in 17 ml of acetone was used in place of glutaric anhydrideas the amine modifier.

The collagen solution was colorless and transparent as determined byboth visual inspection and light/optical microscopy at 40x.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution⁹) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence of the chemically-modified collagen made according to thisexample with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65-95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 7

Chemically-modified collagen was prepared according to the procedure ofExample 3 except that sebacoyl chloride (0.2033 g) in 17 ml of N-methylpyrrolidone was used in place of terephthaloyl chloride as the couplerand succinic anhydride (0.1063 g) in 17 ml of N-methyl pyrrolidone wasused in place of glutaric anhydride as the amine modifier.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution⁹) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence or superiority of the chemically-modified collagen madeaccording to this example with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 73 percent of its initial viscosity within7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 8

Chemically-modified collagen was prepared according to the procedure ofExample 3 except that 4-[p-(o-chlorocarbonyl benzoyl)phenyl] butyrylchloride (0.2652 g) in 17 ml of acetone was used in place ofterephthaloyl chloride as the coupler and succinic anhydride (0.3933 g)in 17 ml of acetone was used in place of glutaric anhydride as the aminemodifier.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution¹⁰) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence of the chemically-modified collagen made according to thisexample with air, BSS™ Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 9

Chemically-modified collagen was prepared according to the procedure ofExample 3 except that diglycolic diacid chloride (0.1442 g) in 17 ml ofacetone was used in place of terephthaloyl chloride as the coupler anddiglycolic anhydride (0.1973 g) in 17 ml of acetone was used in place ofglutaric anhydride as the amine modifier.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution¹⁰) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence or superiority of the chemically-modified collagen madeaccording to this example with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 10

Purified Type I collagen, prepared as in Example 3 (1700 ml of a 0.15percent wt/wt collagen solution in 0.05M acetic acid), was treated at 4°C. with lON sodium hydroxide to raise the pH to 5.0. A solution ofbicyclo-(2,2,2)-oct-7-ene-2,3,5,6-tetracarboxylic-2,3,5,6-dianhydride(0.0810 g) and 1,2,4-benzenetricarboxylic anhydride (0.0660 g) in 17 mlof N,N-dimethylformamide was added all at once to the purified Type Icollagen with stirring. The pH was immediately increased to 9.4 usinglON sodium hydroxide, and the solution was stirred for 10 minutes whilemaintaining the pH at 9.4 by gradual addition of lN sodium hydroxide.After 10 minutes the pH was increased to 12.1 by addition of lON sodiumhydroxide, and the pH was maintained at 12.1 for 2 minutes. The pH wasthen decreased to 9.0 by addition of 6N HCl and maintained at this pHfor 10 minutes while the temperature of the solution was increased from4° C. to 32° C. To this coupled collagen product1,2,4-benzenetricarboxylic anhydride (0.2472 g) in 17 ml acetone wasadded dropwise, along with lN sodium hydroxide to maintain the pH at9.0±0.25. After addition was complete the solution was agitated for 10minutes. The pH was increased to 12.5 for 3 minutes and decreased to 9.0using l0N NaOH and 6N HCl, respectively. After 10 minutes of agitationat pH 9.0, the pH was reduced to 3.2 and the chemically-modifiedcollagen product precipitated. The solution was agitated mildly for 15minutes to ensure complete precipitation. The material was collected bycentrifugation and the collected precipitate was washed successivelyfour times with sterile water at a dilution of 10 parts water to 1 partwet precipitate.

A 2 percent by weight solution of the collagen in BSS™ was colorless andtransparent as determined by both visual inspection and light/opticalmicroscopy at 40_(x).

A 2 percent by weight solution of the collagen in BSS™) was evaluated asan anterior chamber implant in New Zealand white rabbits using theMcDonald-Shadduck system in accordance with the procedure described inExample 1. Such evaluation indicated overall equivalence or superiorityof the chemically-modified collagen made according to this example withair, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 11

Chemically-modified collagen was prepared according to the procedure ofExample 10 except that 1,3,5-benzenetrisulfonyl chloride (0.0727 g) in17 ml N-methyl-pyrrolidone was used in place ofbicyclo-(2,2,2)-oct-7-ene-2,3,5,6-tetracarboxylic-2,3,5,6-dianhydride asthe coupler, and o-sulfobenzoic acid cyclic anhydride (0.0718 g) in 17ml acetone was used in place of 1,2,4-benzenetricarboxylic anhydride asthe amine modifier.

A 2 percent by weight solution of the collagen in BSS™ was colorless andtransparent as determined by both visual inspection and light/opticalmicroscopy at 40x.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution¹¹) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence of the chemically-modified collagen made according to thisexample with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 75 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 35 to 42° C.

EXAMPLE 12

Chemically-modified collagen was prepared according to the method ofExample 10 except that 3,3',4,4'-benzophenonetetracarboxylic dianhydride(0.10 g) was used in place ofbicyclo-(2,2,2)-oct-7-ene-2,3,5,6-tetracarboxylic dianhydride as thecoupling agent.

A 2 percent by weight solution of the collagen in BSS™ was colorless andtransparent as determined by both visual inspection and light/opticalmicroscopy at 40x.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution¹¹) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence or superiority of the chemically-modified collagen madeaccording to this example with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 13

Chemically-modified collagen was prepared according to the procedure ofExample 10 except that 1,2,4,5-benzenetetracarboxylic dianhydride(0.0203 g) was used in place ofbicyclo-(2,2,2)-oct-7-ene-2,3,5,6-tetracarboxylic-2,3,5,6-dianhydride asthe coupling agent, and only 0.1674 g of the amine modifierbenzenetricarboxylic anhydride was used.

A 2 percent by weight solution of the collagen in BSS™ was colorless andtransparent as determined by both visual inspection and light/opticalmicroscopy at 40x.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution¹²) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence or superiority of the chemically-modified collagen madeaccording to this example with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 14

Chemically-modified collagen was prepared according to the procedure ofExample 12 except that succinic anhydride (0.1 g) was used in place ofbenzenetricarboxylic anhydride as the amine modifier.

A 2 percent by weight solution of the collagen in BSS™ was colorless andtransparent as determined by both visual inspection and light/opticalmicroscopy at 40x.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution¹³) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence of superiority of the chemically-modified collagen madeaccording to this example with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 15

Purified Type I collagen, prepared as in Example 1, is dissolved inenough 0.1M acetic acid to provide a 0.15 percent wt/wt solution. Thecollagen solution (300 ml) is chilled to 4° C. and the pH is adjusted to9.0 with lON NaOH. To the vigorously stirring collagen is added1,3-benzenedisulfonyl chloride (0.07 g) dissolved in 3 ml ethyleneglycol dimethyl ether. The pH is maintained at 9.0±0.25 for 15 minutesby gradual addition of lN NaOH.

After 15 minutes, a solution containing 1,2,4-benzenetricarboxylicanhydride (0.05 g) dissolved in 3 ml ethylene glycol dimethyl ether isadded to the collagen solution all at once. The pH is maintained at9.0±0.25 for a period of 45 minutes. The pH is increased to 12.0 for 3minutes by addition of lON NaOH. The pH is then reduced to 3.3 using 6NHCl to precipitate the chemically-modified collagen product. Theprecipitate is collected by filtration and washed using deionized water.The precipitate is reconstituted in a phosphate-buffered solution¹⁴ toprovide a 2 percent wt/wt solution.

The collagen solution is colorless and transparent as determined by bothvisual inspection and light/optical microscopy at 40x.

The collagen solution is evaluated as an anterior chamber implant in NewZealand white rabbits using the McDonald-Shadduck system in accordancewith the procedure described in Example 1. Such evaluation indicatesoverall equivalence of the chemically-modified collagen made accordingto this example with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 16

Purified Type I collagen (750 ml), prepared according to Example 3, wastreated at 4° C. with lON NaOH to raise the pH to 5.0.p-Fluorosulfonylbenzenesulfonyl chloride (0.0251 g) and 0.0124 gglutaric anhydride in 7.5 ml of acetone was added all at once to thevigorously agitating collagen solution. The pH was immediately increasedto 11.7 and then decreased to 9.2 by addition of lON NaOH and 6N HCl,respectively. The solution was agitated for 15 minutes to produce thecoupled collagen product.

The coupled collagen solution was treated at 4° C. with the dropwiseaddition of glutaric anhydride (0.0642 g) in 7.5 ml of acetone, and thepH was maintained at 9.0±0.25 by addition of lH NaOH. After addition wascomplete the solution was agitated for 10 minutes. The pH was reduced to4.0 using 6N HCl, and the solution was agitated for another 10 minutes.The modified collagen precipitate was collected by centrifugation andwashed four times with sterile water at a dilution of 10 parts water to1 part wet precipitate.

A 2 percent by weight solution of the collagen in BSS™ was colorless andtransparent as determined by both visual inspection and light/opticalmicroscopy at 40x.

The collagen solution 2 percent by weight in BSS™) was evaluated as ananterior chamber implant in New Zealand white rabbits using theMcDonald-Shadduck system in accordance with the procedure described inExample 1. Such evaluation indicated overall equivalence or superiorityof the chemically-modified collagen made according to this example withair, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collage solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 17

Purified Type I collagen, as prepared in Example 1, was reconstituted in300 ml of a 0.1M acetic acid solution to provide a 0.20 percent wt/wtsolution. The collagen solution (300 ml) was chilled to 4° C. and the pHwas adjusted to 8.0 with lON NaOH. To the stirring collagen solution wasgradually added 5-chlorosulfonyl-o-anisic acid chloride (0.030 g)dissolved in 3 ml acetone, while maintaining the pH at 8.0 by additionof lN NaOH. A reaction pH of 8.0 was used in order to reduce theconcentration of available free amines thereby controlling the extent ofcoupling. After 6 minutes of reaction the pH was increased to 13 byaddition of 5N NaOH in order to hydrolyze any remaining coupler and stopthe reaction. The pH was maintained at 13 for 2 minutes and then reducedto 9.0 using 6N HCl.

A solution containing glutaric anhydride (0.034 g) dissolved in 3 mlacetone was added to the collagen solution all at once. The pH wasmaintained at 9.0±0.25 by gradual addition of lN NaOH for a period of 30minutes. The pH was then reduced to 4.0 using 6N HCl to precipitate thechemically-modified collagen product. The precipitate was collected andwashed according to the method described in Example 1. The collagenprecipitate was dissolved in balance salt solution (BSS™) to provide a2.0 percent wt/wt solution. The pH was then adjusted to 7.1 using lNNaOH.

The collagen solution (2 percent by weight in a phosphate-bufferedsolution¹⁵) is evaluated as an anterior chamber implant in New Zealandwhite rabbits using the McDonald-Shadduck system in accordance with theprocedure described in Example 1. Such evaluation indicates overallequivalence of the chemically-modified collagen made according to thisexample with air, BSS™ and Healon™.

The collagen solution (about 2 percent by weight in BSS™) exhibits theWeissenberg Effect when tested in accordance with the proceduredescribed in Example 1. The collagen solution is pseudoplastic,exhibiting decreasing viscosities at increasing shear rates when testedin accordance with the procedure of Example 1. The collagen solution isthixotropic, recovering about 65 to 95 percent of its initial viscositywithin 7 minutes of shearing at 0.1 sec⁻¹.

The melt temperature of the 2 percent collagen solution as determined byDifferential Scanning Calorimetry is about 34 to 37° C.

EXAMPLE 18

A cross-linked and further modified collagen was prepared by theaddition of a chemical cross-linking agent and subsequently anisocyanate amine modifier. Procedures were performed using aseptictechnique in a laminar flow hood, which had previously been disinfectedand validated. To a 1000 ml flask serving as the reaction, vessel 500 mlchilled (4 degrees C) Vitrogen 100 ™ brand collagen Type I solution (3mg collagen/ml solution) (Collagen Corp., Palo Alto, CA) were added. ThepH of the solution was brought to 9.0 by the addition of 5N sodiumhydroxide. While the temperature was between 4 and 8 degrees C., thesolution was vigorously agitated using a magnetic stirrer, and 0.28grams of succinyl chloride, which had been kept dry prior to use, wereadded. The reaction was allowed to proceed for 20 minutes whilemaintaining the pH between 9.0 and 9.35 by adding a lN sodium hydroxidesolution as needed.

The cross-linked product obtained was further modified with a reagentwhich will react and covalently bind to the exposed amine groups on thecross-linked collagen molecules. With continued stirring, 0.35 grams ofdry butyl isocyanate (C. Aldrich, Milwaukee, WI) was added to thereaction vessel. The reaction was allowed to proceed for 1 hour atambient temperatures while maintaining the pH between 9.0 and 9.25 byadding lN sodium hydroxide solution as needed. To precipitate out themodified collagen 6N hydrochloric acid was added until the cloudiness ofthe solution stopped increasing as judged visually, and mixing continuedfor 5 minutes. The pH of the solution at this point is about 4.5-4.7.

The thus obtained modified collagen slurry was centrifuged in sterilepolycarbonate tubes using a Sorvall model RC-2 temperature controlledcentrifuge (Dupont Company, Clinical and Instruments Division,Wilmington, Delaware) at 4° C. and 10.000 xg (as measured at the bottomof the tube), for 10 minutes. After disinfecting the tube exteriors withisopropyl alcohol and allowing the alcohol to evaporate under thelaminar flow hood, the clear solution supernatant was decanted leavingthe white collagen precipitate.

The precipitate was washed by mixing with sterile, pyrogen-free water inthe centrifuge tubes, adjusting the pH, if necessary, between 4.5 and4.7, and again centrifuging at 4 degrees C. and 10,000 xg as measured atthe bottom of the tube for 10 minutes. The tubes were again disinfectedwith isopropyl alcohol and dried under the laminar flow hood, and theclear supernatant decanted from the collagen. The washing cycle wasrepeated three more times. The resulting material is substantiallyinsoluble at pH 7 and 37° C., which is determined by the cloudiness ofthe contents and the inability to filter the material when resuspendedin deionized water or physiological saline.

A portion of the modified collagen is suspended in physiological salineat 1.5% by weight total solids. The suspension is useful in soft tissueaugmentation. An identical suspension is prepared and freeze-dried. Thefreeze-dried product is useful as a surgical sponge.

What is claimed is:
 1. A chemically-modified collagen compoundcomprising at least two native collagen molecules coupled at least onelysine epsilon amino group present on each of said collagen molecules bya coupling group, said coupling group comprising at least two moietiesselected from the group consisting of carbonyl and sulfonyl groups,wherein non-coupled lysine epsilon amino groups are linked to aminemodifying groups selected from groups of the formula ##STR8## wherein Ris a substituted or unsubstituted C₁₋₈ aliphatic, C₅₋₈ alicyclic, orC₆₋₁₀ aromatic group having 0-5 heteoatoms, and the formula ##STR9## andits metal salts.
 2. The compound of claim 1 wherein at least 60% of thenon-coupled lysine epsilon amino groups are linked to the aminemodifying groups.
 3. The compound of claim 1 wherein the amine modifyinggroups are groups of the formula ##STR10## wherein R is a substituted orunsubstituted C₁₋₈ aliphatic, C₅₋₈ alicyclic, or C₆₋₁₀ aromatic grouphaving 0-5 heteroatoms.
 4. The compound of claim 3 wherein R is C₁₋₈alkyl, C₅₋₈ cycloalkyl, C₆₋₁₀ aryl, C₆₋₁₀ substituted aryl, or C₆₋₁₀aralkyl.
 5. The compound of claim 4 wherein R is butyl.
 6. The compoundof claim 1 wherein the amine modifying groups are groups of the formula##STR11## or its metal salts.
 7. A collagen fraction comprised ofcollagen molecules, about 10-80% of which comprises the compound ofclaim
 6. 8. A viscoelastic collagen solution comprising about 0.5-5.0%by weight of the collagen fraction of claim 7 dissolved in aphysiological buffer solution.
 9. A viscoelastic collagen solutioncomprising about 0.5 to 5.0 percent by weight of the collagen compoundof claim 6 dissolved in a physiological buffer solution, wherein thecoupling group is present in said chemically-modified collagen compoundto such an extent that a 2 percent by weight solution of said collagencompound in said physiological buffer has a melt temperature of betweenabout 32° C. and 48° C.
 10. A collagen fraction comprised of collagenmolecules, about 10-80% of which comprise the compound of claim
 1. 11. Achemically modified collagen composition comprising about 0.5-5.0% byweight of the collagen fraction of claim 10 suspended in a physiologicalbuffer solution.
 12. A chemically modified collagen compositioncomprising about 0.5 to 5.0 percent by weight of the collagen compoundof claim 1 suspended in a physiological buffer solution.
 13. Thechemically modified collagen composition of claim 12 wherein thecoupling group is present in said chemically-modified collagen compoundto such an extent that a 2 percent by weight suspension of said collagencompound in said physiological buffer has a melt temperature of betweenabout 40° C. and 70° C.
 14. The chemically modified collagen compositionof claim 12 wherein said coupling group is --CO(CH₂)₂ --CO--or--CO(CH₂)₃ --CO--, and said modifying group is --CONH(CH₂)₃ CH₃. 15.The chemically modified collagen composition of claim 12 wherein saidcoupling group is --CO(CH₂)₃ --CO-- or--CO(CH₂)₂ --CO--, and saidmodifying group is --CHCOOH--CHOH--COOH.
 16. A method of making achemically-modified collagen compound comprising the steps of(a) addingto an aqueous solution of at least about 0.05 percent by weight nativecollagen, a coupling agent in an amount equal to about 1 to 600 moles ofcoupling agent per mole of native collagen, said coupling agent selectedfrom the group consisting of di- and tri-carboxylic acid halides, di-and tri-sulfonyl halides, di- and tri-anhydrides, di- and tri-reactiveactive esters, and compounds having at least two moieties selected fromthe group consisting of carboxylic acid halide, sulfonyl halide,anhydride and active ester; (b) reacting said native collagen and saidcoupling agent at a pH of above about 8 and at a temperature of betweenabout 0 and 35° C., for a time period sufficient to either react orhydrolyze substantially all of said coupling agent; (c) adding to saidreaction mixture a mono-reactive amine-modifying agent, saidamine-modifying agent being selected from agents of the formula

    O═C═N--R

wherein R is a substituted or unsubstituted C₁₋₈ aliphatic, C₅₋₈alicyclic, or C₆₋₁₀ aromatic group having 0-5 heteroatoms, and epoxysuccinic acid in an amount equal to at least about 100 moles ofamine-modifying agent per mole of native collagen; and (d) conductingthe amine-modification reaction at a pH of greater than about 8 and at atemperature of between about 0 and 35° C., for a time period sufficientto either react or hydrolyze substantially all of said amine-modifyingagent
 17. The method of claim 16 wherein said coupling agent is selectedfrom the group consisting of terephthaloyl chloride;bicyclo-(2,2,)-oct-7 -ene-2,3,5,6-tetracarboxylic-2,3,5,6-dianhydride;1,2,4,5-benzenetetracarboxylic dianhydride;p-fluorosulfonylbenzenesulfonyl chloride; and wherein the pH of thereaction mixture is initially maintained at about 3 to about 5.5 beforeraising the pH to above about
 8. 18. A method of making achemically-modified collagen compound comprising the steps of:(a) addingto an aqueous solution of at least about 0.05 percent by weight nativecollagen, a coupling agent in an amount equal to about 1 to 600 moles ofcoupling agent per mole of native collagen and a mono-reactiveamine-modifying agent in an amount equal to at least about 50 moles ofamine-modifying agent per mole of native collagen; said coupling agentselected from the group consisting of di- and tri-carboxylic acidhalides, di- and tri-sulfonyl halides, di- and tri-anhydrides, di- andtri-reactive active esters and compounds having at least two moietiesselected from the group consisting of carboxylic acid halide, sulfonylhalide, anhydride and active ester; and wherein said amine-modifyingagent is selected from groups of the formula

    O═C═N--R

wherein R is a substituted or unsubstituted C₁₋₈ aliphatic, C₅₋₈alicyclic, or C₆₋₁₀ aromatic group having 0-5 heteroatoms, and epoxysuccinic acid; (b) allowing the reaction mixture to react at a pH ofgreater than about 8 and at a temperature of between about 0 and 35° C.,for a time period sufficient to either react or hydrolyze substantiallyall of said coupling agent and said amine-modifying agent; (c) adding tothe reaction mixture of step (b) additional mono-reactiveamine-modifying agent in an amount equal to at least about 50 moles ofmodifying agent per mole of native collagen; and (d) allowing thereaction mixture of step (c) to react at a pH of greater than about 8and at a temperature of between 0 and 35° C., for a period of timesufficient to either react or hydrolyze substantially all of saidmodifying agent.
 19. A method of making a chemically-modified collagencompound comprising the steps of:(a) adding to an aqueous solution of atleast about 0.05 percent by weight native collagen a mono-reactiveamine-modifying agent in an amount equal to at least about 25 moles ofamine-modifying agent per mole of native collagen, said amine-modifyingagent being selected from groups of the formula

    O═C═N--R

wherein R is a substituted or unsubstituted C₁₋₈ aliphatic, C₅₋₈alicyclic, or C₆₋₁₀ aromatic group having 0-5 heteroatoms, and epoxysuccinic acid; (b) allowing the reaction mixture of step (a) to react ata pH of greater than about 8 and at a temperature of about 0 to 35° C.for a period of time sufficient to either react or hydrolyzesubstantially all of said amine-modifying agent; (c) adding to themixture produced in step (b) a coupling agent in an amount equal toabout 1 to 600 moles of coupling agent selected from the groupconsisting of di- and tri-carboxylic acid halides, di- and tri-sulfonylhalides, di- and tri-anhydrides and di- and tri-reactive active esters,and compounds having at least two moieties selected from the groupconsisting of carboxylic acid halide, sulfonyl halide, anhydride andactive ester; and (d) allowing the reaction mixture of step (c) to reactat a pH of greater than about 8 and at a temperature of about 0 to 35°C. for a time period sufficient to either react or hydrolyzesubstantially all of said coupling agent; (e) adding to the reactionmixture of step (d) additional amine-modifying agent in a concentrationof at least about 75 moles of modifying agent per mole of nativecollagen; and (f) allowing the reaction mixture of step (d) to react ata pH of greater than about 8, and at a temperature of between about 0and 35° C., for a period of time sufficient to either react or hydrolyzesubstantially all of said amine-modifying agent.
 20. A method ofachieving hemostasis comprising placing an effective amount of thechemically-modified collagen compound of claim 1 in contact with ableeding wound.
 21. A method of augmenting soft tissue comprisingplacing an effective amount of the chemically-modified collagencomposition of claim 11 in contact with the augmentation site.